Oncolytic viruses represent a valuable new modality for the treatment of malignant diseases such as breast cancer, as they can selectively replicate in tumor cells while sparing normal cells. Unlike typical gene-based forms of cancer therapy, oncolytic viruses kill tumor cells directly through their selective replication/cytolysis and consequent spread to surrounding tumor tissues. These properties offer a major advantage over the inherent inefficiency of gene delivery and thus the limited tumor cell killing often seen with gene-mediated therapy. Work conducted during the previous funding period has demonstrated that incorporation of cell membrane fusion activity into an oncolytic herpes simplex virus (HSV) can significantly enhance the tumor killing effect of the virus against breast cancer. Moreover, tumor destruction by a doubly fusogenic oncolytic HSV (Synco-2D) can elicit effective antitumor immune responses. The current proposal builds upon these prior advances and investigates strategies that can maximize the antitumor effect of Synco-2D, thus increasing the likelihood of its clinical success. The Aim 1 of our proposed studies will examine if the antitumor activity of Synco-2D can be further enhanced by rationally designed combinatorial strategies, including incorporation of a thrombosis-inducing factor into the virus and alteration of tumor microenvironment to favor the virus replication. Aim 2 will explore strategies that can improve the functionality of antitumor immune responses elicited by Synco-2D-mediated oncolysis. In particular, we will investigate if epigenetic upregulation of costimulatory molecules can significantly improve the functionality of the induced antitumor immunity. We will also determine if the acidic tumor microenvironment affects the antitumor activity of immune effector cells, and explore strategies that can reverse the adverse effect. Aim 3 will seek to develop strategies that enable Synco-2D to destroy tumor stem cells for a long-lasting therapeutic effect. The long-term goal of this project is to develop a potent virotherapy capable of producing significant therapeutic effects against advanced-stage breast cancer, with the potential to be translated into clinical use in the near future. PUBLIC HEALTH RELEVANCE: Despite recent progresses in early diagnosis and treatment, patients with breast cancer still have very poor prognoses. This proposal seeks to develop a novel virotherapy to treat patients with late stage breast cancer and success of the proposed studies will greatly benefit patients suffering from this malignant disease.